Devices for delivering agents and methods of using thereof

Abstract

Devices for delivering agents including a microelectrode array are described herein. Methods of using such devices for delivering agents via electroporation are also described. The devices are useful for various applications, such as medical treatments.

Description

[0001] DEVICES FOR DELIVERING AGENTS AND METHODS OF USING THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] The present application claims priority to U.S. Provisional Application No. 63 / 599,188, filed November 15, 2023, the contents of which is hereby incorporated by reference in its entirety.

[0004] FIELD OF THE INVENTION

[0005] The invention is generally in the field of devices for delivery of one or more agents and uses thereof.

[0006] BACKGROUND OF THE INVENTION

[0007] Various types of devices have been investigated for delivery of agents, such as drugs, nucleic acids, and biologicals through tissues, such as skin, and into cells. However, the stratum comeum of mammalian skin is an effective barrier, particularly for molecules larger than 500 Da. Yet another challenge is how to effectively facilitate the uptake of drugs into cells in the skin.

[0008] Currently known conventional devices using ultrasound and / or electroporation usually require costly technology, and in all cases require access to a continuous source of electric power.

[0009] Accordingly, there remains a need for devices which can be used for administration of agents which can address the above-referenced limitations and issues.

[0010] Therefore, it is the object of the present invention to provide devices for delivery of agents to, for instance, a tissue.

[0011] It is a further object of the present invention to provide methods of using such devices.

[0012] It is still a further object of the present invention to provide kits containing such devices and / or components thereof.

[0013] SUMMARY OF THE INVENTION

[0014] Devices for delivering agents and methods of using thereof are described herein. In one non-limiting instance, such a device includes: a piezoelectric pulse generator; an array of biocompatible and conductive microelectrodes electrically coupled to the piezoelectric pulse generator; and a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses; wherein the array of biocompatible and conductive microelectrodes comprises an opening for insertion of at least one needle therein for delivery of at least one agent; wherein a syringe comprising a container for holding the at least one agent can be coupled to the at least one needle and the at least one agent can be administered into a biological tissue by actuation of the syringe; wherein insertion of the array of biocompatible and conductive microelectrodes into the biological tissue and activation of the piezoelectric pulse generator generates the one or more electrical pulses through the array of biocompatible and conductive microelectrodes to electroporate cells in the biological tissue to permit administration of the at least one agent into the electroporated cells.

[0015] The device can be applied to any suitable biological tissue(s) of a subject. That is, the electrical pulse and one or more agents may be delivered to the tissue, and cells thereof, that are at or near a physically accessible part of the body of the subject. The delivery of electrical pulses and at least one agent may be to cells in the skin, cells in epithelial layers of the body, or cells in the body’s interior that are accessible for example laparoscopically or due to a surgical intervention. In some instances, the biological tissue comprises mammalian skin. In some instances, the target tissue site on which the device is used includes the dermis or epidermis. In some other instances, the biological tissue includes a mucosal membrane. A mucosal membrane, also known as a mucous membrane, is a type of epithelial tissue that lines various cavities and structures within the body that are exposed to the external environment. In some cases, the target tissue site may be in the mouth, nose, eye, gastrointestinal tract, or vagina.

[0016] Such devices can be used for various medical applications. In one non-limiting instance, the device may be used in a method of delivering an agent into or across a biological tissue, the method including the steps of:

[0017] (a) positioning a device adjacent to a biological tissue site, the device including: a piezoelectric pulse generator; an array of biocompatible and conductive microelectrodes electrically coupled to the piezoelectric pulse generator; a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses; a syringe comprising a container holding at least one agent; wherein the array of biocompatible and conductive microelectrodes includes at least one needle for delivery of the at least one agent embedded therein; wherein the syringe is coupled to the at least one needle for administering the at least one agent into a biological tissue by actuation of the syringe;

[0018] (b) contacting the array of biocompatible and conductive microelectrodes including the at least one needle embedded therein into the target biological tissue site;

[0019] (c) activating the switch to generate and deliver one or more electrical pulses through the array of biocompatible and conductive microelectrodes into the target biological tissue site to electroporate cells at the target biological tissue site; and

[0020] (d) administering the at least one agent into the target biological tissue site through the at least one needle by actuation of the syringe.

[0021] Typically, the methods include manually applying a force to the device effective to penetrate a biological tissue site surface with the array of biocompatible and conductive microelectrodes, and then manually pressing the switch on the device that triggers a mechanical force onto a piezoelectric pulse generator to produce one or more electric pulses effective to induce electroporation (z.e., pore formation) in the cells of the biological tissue site. Depressing the plunger of the syringe can actuate agent release, from the syringe container.

[0022] The devices and methods described can be used to deliver at least one agent into or across a biological tissue. The devices and methods described can be used to deliver agents known to those skilled in the art. In some instances, the at least one agent can be selected from a nucleic acid (such as DNA and / or RNA, nucleic acid encoding a vaccine, nucleic acid encoding a therapeutic protein, nucleic acid encoding a hormone, or a nucleic acid encoding a monoclonal antibody), a DNA-plasmid, a minicircle DNA plasmid, an mRNA, a self- amplifying RNA, a circular RNA, a transfer RNA, a chemotherapeutic agent, a biologic, a prophylactic, a DNA- launched self-amplifying RNA, a DNA-launched virus, a linear DNA, and combinations thereof. In some instances, the at least one agent is a therapeutic, wherein the therapeutic is a RNA therapeutic or DNA therapeutic.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A shows a non- limiting illustrative view of a device 100 having a needle component 110, a syringe 120, a syringe plunger 125, a device casing 130, an optional finger rest 140, an optional screw 150 for holding the syringe when placed into the device, an array of biocompatible and conductive microelectrodes 200, a piezoelectric pulse generator 300, and a mechanical switch 310 for activating the piezoelectric pulse generator.

[0025] Figure IB shows a non-limiting illustrative head-on view of a device 100 having a needle component 110, a syringe 120, a syringe plunger 125, a device casing 130, an optional finger rest 140, an optional screw 150 for holding the syringe when placed into the device, and an array of biocompatible and conductive microelectrodes 200.

[0026] Figure 1C shows a non-limiting illustrative side view of a device 100 having a needle component 110, a syringe 120, a syringe plunger 125, a device casing 130, an optional finger rest 140, an optional screw 150 for holding the syringe when placed into the device, an array of biocompatible and conductive microelectrodes 200, a piezoelectric pulse generator 300, and a switch 310 for activating the piezoelectric pulse generator.

[0027] Figure 2A shows a non-limiting illustration of an MEA 200 having an array of biocompatible and conductive microelectrodes 210 see Figure 2B), an opening for at least one needle component therein 220, and a base 230.

[0028] Figure 2B shows a non-limiting illustration of an array of biocompatible and conductive microelectrodes 210.

[0029] Figure 2C shows a non-limiting illustration of a needle component 110 having a needle end tip.

[0030] Figure 3 A shows a non- limiting illustration of a needle component 110 placed into base 230 through the opening / hole such that the needle tip of the needle component 110 is exposed adjacent to the microelectrodes of the array 210.

[0031] Figure 3B shows a non-limiting illustration of a needle component 110 placed into base 230 through the opening / hole such that the needle tip of the needle component 110 is exposed adjacent to the microelectrodes of the array 210.

[0032] Figure 4 shows a non-limiting illustration of a piezoelectric pulse generator 300 and a switch 310, which is coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate electrical pulse(s).

[0033] Figure 5 A shows a non- limiting illustration of a syringe 120 having a container 126, which can hold one or more agents, a plunger 125, which can be used to administer the agents when actuated, and a delivery end 127 which optionally includes an attachment or lock, which can be used for connecting and attaching a needle component.

[0034] Figure 5B shows a non-limiting illustration of a syringe 120 coupled to a needle component 110 and an MEA 200. Figures 6A and 6B are representative images of an ex vivo human skin sample taken right after administration (Figure 6A) and the corresponding IVIS image taken 24 hours after administration, demonstrating signals for: Naive, ID, and VID experimental groups (Figure 6B).

[0035] Figures 7A-7D are representative IVIS images taken 24 hours after administration, demonstrating signals for: VID injections using different drug delivery needle lengths (i.e., 1.0 mm, 1.1 mm, 1.25 mm, 1.3 mm) (Figure 7A), Naive, ID, VID, and VIDEP experimental groups (Figure 7B), ID, VID, and VIDEP experimental groups (Figure 7C), and Naive, VID, VIDEP, and ID BTX experimental groups (Figure 7D).

[0036] Figures 8A and 8B are bar graphs showing DNA-Luc delivery using Piezopen augments gene expression compared to ID BTX and (V)ID injections alone 1 day post-administration. Plots of total flux (Figure 8 A) and average radiance (Figure 8B) are shown for: (1) Naive, (2) ID injection of DNA-Luc, (3) VID injection of DNA-Luc, (4) ID BTX, and (5) VIDEP using PiezoPen experimental groups. NNatve = 6, NID = 11, NVID = 11, NIDBTX= 3, and NVIDEP Separate = 7. Statistics are not included but an ordinary one-way ANOVA with Tukey’s multiple comparisons test was performed.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] Devices for delivering agents and methods of using thereof are described below. In some cases, the devices are, for instance, useful for delivery of agents larger than 500 Da.

[0039] I. Definitions

[0040] The term “conductive” refers to materials that allow for the flow of electric current with low or minimal resistance. Such materials can be made of or contain one or more metals or can be made of other materials, such as conductive polymers / plastics.

[0041] The terms “individual,” “subject,” and “patient” are used interchangeably, and refer to a mammal, including, but not limited to, humans, rodents, such as mice and rats, and other laboratory animals.

[0042] The term “subject” refers to either a human or non-human animal.

[0043] The term “biocompatible” refers to one or more materials that are neither themselves toxic to a subject (e.g., an animal or human), nor degrade at a rate that produces subunits or other by-products at toxic concentrations in the subject.

[0044] The term “treating” refers to inhibiting, ameliorating, impeding, alleviating, or relieving a disease, disorder, or condition from occurring in a subject or causing regression of the disease, disorder, and / or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected. The term “nucleic acid” refers to any natural or synthetic linear and sequential arrays of nucleotides and nucleosides, for example, DNA including complementary DNA (cDNA), plasmid DNA (pDNA), circular DNA (cDNA), mini-circle DNA (mDNA), replicating RNA (repRNA), messenger RNA (mRNA), small interfering RNA (siRNA), transfer RNA (tRNA), microRNA (miRNA), guide strand RNA (sgRNA), polynucleotides, oligo-nucleotides, oligonucleosides and derivatives thereof. Such nucleic acids may be collectively referred to as “constructs,” or “plasmids”.

[0045] The term “electroporation,” as used herein, refers to the application of an electric field to cells present in a tissue, which makes their cell membranes temporarily permeable to allow the entry of agents that typically cannot cross or easily cross the cell membrane. Temporary pores are created by the electric field which allows for the entry of agents, such as DNA, RNA, proteins, drugs, or molecules into the cells.

[0046] The terms “RNA vaccine,” or “messenger RNA (mRNA) vaccine” can be used interchangeably and refer to a type of vaccine that utilizes a small piece of genetic material from the virus or pathogen against which protection is desired by introducing the viral mRNA into a subject, prompting the cells to produce a harmless piece of the virus (usually a viral protein) that triggers an immune response. This immune response helps the subject recognize and fight the actual virus if the subject is later exposed to it.

[0047] The term “DNA vaccine” refers to a type of vaccine that uses a small, circular piece of DNA, usually derived from a virus or bacterium, to stimulate an immune response in a subject. DNA vaccines do not contain the whole pathogen but instead carry only specific genes that code for antigens — proteins or pieces of proteins that the immune system recognizes as foreign. The DNA is typically introduced into the cells of the vaccine recipient, where the host cells use the instructions encoded in the DNA to produce the antigen, which triggers an immune response, including the production of antibodies and the activation of immune cells. If the subject is later exposed to the actual pathogen, their immune system is prepared to recognize and combat the pathogen.

[0048] The term “electric field strength,” as used herein refers to nominal electrical field strength, which is the voltage divided by the distance between electrodes of opposite charge.

[0049] The term “peak voltage,” as used herein, refers to the maximum voltage drop achieved across electrodes when applied to biological tissue; the term “peak-to-peak voltage” refers to the voltage difference between the peak positive voltage and the peak negative voltage when applied to biological tissue; the term “peak static voltage” refers to the maximum voltage drop achieved across the electrodes when the electrodes are directly applied to the leads of an oscilloscope; the term “peak-to-peak static voltage” refers to the voltage difference between the peak positive voltage and the peak negative voltage when electrodes are directly applied to the leads of an oscilloscope; the term “peak current” refers to the maximum current through the biological tissue; the term “nominal electric field strength” refers to the voltage across the electrodes divided by the spacing between oppositely charged electrodes; the term “initial pulse length” refer to the time between the initiation of the pulse and the time when the voltage first returns to the voltage before initiation of the pulse when applied to biological tissue; and the term “total pulse length” refers to the time between the initiation of the pulse and the time when the voltage no longer achieves an absolute value greater than 10% of the absolute value of the peak voltage when applied to biological tissue.

[0050] Numerical ranges disclosed in the present application include, but are not limited to, ranges of voltages, ranges of lengths, ranges of integers, ranges of times, ranges of areas, etc. The disclosed ranges, of any type, disclose individually each possible number that such a range could reasonably encompass, as well as any sub-ranges and combinations of sub-ranges encompassed therein. For example, disclosure of a voltage range is intended to disclose individually every possible voltage value that such a range could encompass, consistent with the disclosure herein.

[0051] Use of the term “about” is intended to describe values either above or below the stated value, which the term “about” modifies, in a range of approx. + / - 10%; in other instances, the values may range in value either above or below the stated value in a range of approx. + / - 5%. When the term “about” is used before a range of numbers (z.e., about 1-5) or before a series of numbers (z'.e. , about 1, 2, 3, 4, etc.) it is intended to modify both ends of the range of numbers or each of the numbers in the series, unless specified otherwise.

[0052] IL Devices for Delivering Agents and Components Thereof

[0053] Devices which can deliver one or more agents, and which incorporate electroporation are described herein. In one non-limiting instance, such a device includes: a piezoelectric pulse generator; an array of biocompatible and conductive microelectrodes electrically coupled to the piezoelectric pulse generator; and a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses; wherein the array of biocompatible and conductive microelectrodes comprises an opening for insertion of at least one needle therein for delivery of at least one agent; wherein a syringe comprising a container for holding the at least one agent can be coupled to the at least one needle and the at least one agent can be administered into a biological tissue by actuation of the syringe; wherein insertion of the array of biocompatible and conductive microelectrodes into the biological tissue and activation of the piezoelectric pulse generator generates the one or more electrical pulses through the array of biocompatible and conductive microelectrodes to electroporate cells in the biological tissue to permit administration of the at least one agent into the electroporated cells.

[0054] In some instances, the syringe (loaded with an agent(s), such as a drug(s)) is coupled to the device and the device is used to administer the agent(s) intradermally, but without applying electric pulses or toggling the piezoelectric pulse generator.

[0055] Figure 1 A shows a non-limiting illustrative view of a device 100 having a needle 110, a syringe 120, a syringe plunger 125, a device casing 130, an optional finger rest 140, an optional screw 150 for holding the syringe when placed into the device, an array of biocompatible and conductive microelectrodes 200 (also referenced as a microelectrode array “MEA”), a piezoelectric pulse generator 300, and a mechanical switch 310 for activating the piezoelectric pulse generator. Figure IB shows a non- limiting illustrative head-on view of a device 100 having a needle 110, a syringe 120, a syringe plunger 125, a device casing 130, an optional finger rest 140, an optional screw 150 for holding the syringe when placed into the device, and an array of biocompatible and conductive microelectrodes 200. Figure 1C shows a non-limiting illustrative side view of a device 100 having a needle 110, a syringe 120, a syringe plunger 125, a device casing 130, an optional finger rest 140, an optional screw 150 for holding the syringe when placed into the device, an array of biocompatible and conductive microelectrodes 200 (also referenced as a microelectrode array, “MEA”), a piezoelectric pulse generator 300, and a switch 310 for activating the piezoelectric pulse generator.

[0056] In some instances, the array of biocompatible and conductive microelectrodes extends from a base and the housing contains the piezoelectric pulse generator and the switch. In some other instances, the array of biocompatible and conductive microelectrodes extends from a base and the housing contains the base, the piezoelectric pulse generator, and the switch. In some instances, the base and the array of biocompatible and conductive microelectrodes may form part of a cartridge configured to be releasably coupled to the housing that contains the piezoelectric pulse generator. The base and array can form an independent and self-contained MEA unit which can be coupled to the housing of the device by any suitable means, such as with pins or screws. In some instances, the device includes an optional screw that can be used to hold a syringe which is placed into the device, as shown, in Figures 1A-C. In some cases, the screw can be replaced by other holding components, such as pins, clips, and the like to hold the syringe in place.

[0057] In certain instances, the device is designed, sized, and shaped to be handheld and manually operable, such as with a single operator hand. For instance, the device may include an optional finger rest 140, as shown in Figure 1.

[0058] The device typically has no stored electrical energy (e.g., it has no battery) and has no wired or wireless attachment to an energy source (e.g., it is not plugged into an electrical outlet). The energy that generates the voltage across the array microelectrodes of the device is provided by the mechanical action of the user of the device when activating the mechanical switch. For example, the energy associated with manually pressing a switch (or button) on the device provides the energy that triggers a mechanism that generates a voltage across the microelectrodes. The energy generated by the piezoelectric component of the device may transduce the mechanical energy input by the user into electrical energy that generates the voltage.

[0059] The device can be applied to any suitable biological tissue(s) of a subject. That is, the electrical pulse and one or more agents may be delivered to the tissue, and cells thereof, that are at or near a physically accessible part of the body of the subject. The delivery of electrical pulses and at least one agent may be to cells in the skin, cells in epithelial layers of the body, or cells in the body’s interior that are accessible for example laparoscopically or due to a surgical intervention. In some instances, the biological tissue comprises mammalian skin. In some instances, the target tissue site on which the device is used includes the dermis or epidermis. In some other instances, the biological tissue includes a mucosal membrane. A mucosal membrane, also known as a mucous membrane, is a type of epithelial tissue that lines various cavities and structures within the body that are exposed to the external environment. In some cases, the target tissue site may be in the mouth, nose, eye, gastrointestinal tract, or vagina.

[0060] In some instances, the device may weigh under 300 g, under 150 g, under 100 grams, or under 50 grams and may have a total volume of less than about 1000 cm3, less than 750 cm3, less than 500 cm3, less than 250 cm3, less than 100 cm3, less than 50 cm3, or less than 20 cm3, and requires no battery or power sources beyond the piezoelectric crystal of the generator, which is triggered by the mechanical input of a user.

[0061] In some instances, the array of biocompatible and conductive microelectrodes is replaceable and disposable, and / or the piezoelectric pulse generator and the switch are each independently intended for single-use or are intended to be reusable. In certain instances, the array of biocompatible and conductive microelectrodes and syringe may each independently be reusable.

[0062] In some instances, the device may further include a patch and / or a coating, which are each optionally dissolvable. Without limitation, the microelectrodes can be coated with an agent (such as a drug), such that when they are inserted into tissue (such as skin), the coating releases the agent(s) which diffuse into the tissue. This can be achieved, for example, where a coating is formed by dipping the microelectrodes into a solution containing active agent(s) and drying it to form a coating thereon. Various different coating methods are known. For example, see J Pharmacol Exp Ther. 2019 Sep; 370(3): 555-569. In some instances, a dissolvable coating is provided on a separate dissolvable micro-needle patch containing an agent(s) (such as a drug(s)), that can be housed with (such as below) the microelectrode array and is used when the electroporation device is inserted, such that it first causes insertion of the dissolvable microneedle patch which delivers the agent(s) / drug(s), and then insertion of the microelectrode array with triggering is used to administer electrical pulses. In still other instances, the MEA may itself be a dissolvable micro-needle patch, which otherwise meets the requirements of the MEA described.

[0063] A. Components of Devices for Delivering Agents

[0064] The various components of the devices described herein are provided in detail below. i. Microelectrode Array (MEA)

[0065] In one non-limiting instance, a microelectrode array, also referred to as a microneedle electrode array, (MEA) includes: an array of biocompatible and conductive microelectrodes comprising an opening for at least one needle therein; wherein a syringe comprising a container for holding at least one agent can be coupled to the at least one needle for delivery of the at least one agent.

[0066] For the MEAs, the array is typically around the at least one needle that fits through the opening, such that the opening is present through the base of the MEA. In some instances, there is also a possibility for the at least one needle that fits through the opening to be adjacent to the array, such as for injecting through a side port needle.

[0067] In some instances, the microelectrodes of the array are tissue-penetrating microelectrodes such as, microneedle electrodes. In some instances, the MEA does not include flat microelectrodes.

[0068] In some instances, the MEA includes a base from which the array of biocompatible and conductive microelectrodes extends. For instance, Figure 2A shows a non-limiting illustration of an MEA 200 having an array of biocompatible and conductive microelectrodes 210, an opening for at least one needle therein 220, and a base 230.

[0069] Figure 2B shows a non-limiting illustration of an array of biocompatible and conductive microelectrodes 210. In some instances, the biocompatible and conductive electrodes are formed of a metal; or wherein the biocompatible and conductive electrodes are formed of a mixture of epoxy and graphite; or a mixture of glass and platinum; or wherein the biocompatible and conductive electrodes are formed of a conductive ceramic, such as those known in the art. In some instances, the metal is stainless steel. Other suitable metals can include, without limitation, nickel, iron, titanium, copper, silver, gold, magnesium, cobalt, chromium, or other metals or alloys thereof. In some cases, the electrodes can be made of a cobalt-chromium alloy.

[0070] In some instances, the biocompatible and conductive microelectrodes of the array extend from one or more metal plates. The one or more plates can form part of a base and are typically a separate component from the base. In some instances, the one or more plates can be perpendicular to the microelectrode array central axis. In some other instances, the one or more metal plates can be parallel to the microelectrode array central axis. It is understood that on each plate, some of the microelectrodes may be positive and some may be negative. In some instances, a linear array of the biocompatible and conductive microelectrodes extends from one edge of each of the one or more metal plates. In some instances, the one or more plates are parallel to each other and spaced apart from one another. In some other instances, the biocompatible and conductive microelectrodes of the array extend from a single metal plate, and the array forms a two-dimensional array. It is understood that when the microelectrodes of the array extend from a single metal plate there would be at a minimum two metal plates / sheets where one functions as an anode and another as a cathode. In some instances, the biocompatible and conductive microelectrodes of the array extend from one or more metal plates and the array forms a two-dimensional array. All of the microelectrodes on a plate may not be connected to each other. The connections may be selective among the microelectrodes, and there can be multiple connections / electrode groupings. In some instances, the plates are fixed in the base, which acts as an insulating holder, made, for example, of a non-conducting polymeric material. For example, the insulating holder may be a cartridge body having a series of slots into which the plates forming part of the array may be fixed and where the microelectrodes extend from a surface of the cartridge body (z.e., base).

[0071] In still other instances, the biocompatible and conductive microelectrodes of the array extend from at least one non-electrically conductive plate, and electrical connections are provided between the biocompatible and conductive microelectrodes, where the electrical connections can be located on a surface of the at least one non-electrically conductive plate. In such instances, for example, the non-electrically conductive plate and microelectrodes can be part of the same piece, where a single piece of metal can be chem-etched, which forms a base plate from which the microneedle electrodes extend and are associated, joined, or inserted with the non-electrically conductive plate. In some instances, the electrical connections cross from a first side of the at least one non-electrically conductive plate to an opposed second side of the plate through holes in the at least one non-electrically conductive plate.

[0072] In some instances, two or more microelectrodes of the array with the same polarity (z.e., cathodes or anodes) can form part of the same piece of electrically conducting materials (e.g., metal), for example, formed from a single piece of material and cut, etched or otherwise processes to achieve the microelectrode array geometry. The material could be a sheet and the microelectrodes could be in the same plane as the sheet, or they could be at a non- zero angle, such as about 90 degrees, from the plane of the sheet.

[0073] In some instances, the microelectrodes of the array may be formed of a non-electrically conductive material and the array further includes a conductive material e.g., metal) deposited and / or patterned onto the surface of the array in such a way that all of the anodes are electrically connected and all of the cathodes are electrically connected, but the anodes and cathodes and electrically isolated from each other on the microelectrode array. In still other instances, the microelectrodes of the array are made of a conductive material and are connected to a substrate that is not conductive, such that the microelectrodes are electrically connected to each other by patterning conductive material on one or more of the surfaces of the substrate and / or by electrical connections, which may involve wires electrically connected to the microelectrodes via the array surface and / or through holes in the substrate.

[0074] In certain instances, the base forms a cartridge that contains the array of biocompatible and conductive microelectrodes and further includes a first receptacle for mating engagement with a first electrode and a second receptacle for mating engagement with a second electrode, the first and second receptacles being in electrical communication with the microelectrodes of the array.

[0075] In some instances, the biocompatible and conductive microelectrodes of the array define and have an area of between about 1 mm2and 50 cm2or about 1 mm2and 25 cm2, as well as sub-ranges or individual values contained within. The area defined by the biocompatible and conductive microelectrodes of the array refers to the maximum area on which the microelectrodes can form contact with a tissue (such as skin); i.e., skin contact area. In some instances the array of biocompatible and conductive microelectrodes can include from: (1) about 2 to 3000 microelectrodes, about 2 to 2000 microelectrodes, or about 2 to 1000 microelectrodes, as well as sub-ranges or individual values contained within the aforementioned ranges; or (2) at least: about 25 microelectrodes, about 50 microelectrodes, about 100 microelectrodes, about 150 microelectrodes, about 200 microelectrodes, about 250 microelectrodes, about 300 microelectrodes, about 350 microelectrodes, about 400 microelectrodes, about 450 microelectrodes, about 500 microelectrodes, about 550 microelectrodes, about 600 microelectrodes, about 650 microelectrodes, about 700 microelectrodes, about 750 microelectrodes, about 800 microelectrodes, about 850 microelectrodes, about 900 microelectrodes, about 950 microelectrodes, about 1000 microelectrodes, about 1500 microelectrodes, about 2000 microelectrodes, about 2500 microelectrodes, or about 3000 microelectrodes.

[0076] In some instances, the biocompatible and conductive microelectrodes of the array can each independently have a length from 10 pm to 8 mm, as well as sub-ranges or individual values contained within; or the biocompatible and conductive microelectrodes of the array each independently have a length of at least about 500 pm, 600 pm, 650 pm, 700 pm, 750 pm, 800 pm, 900 pm, 950 pm, 1000 pm, 1050 pm, 1100 pm, 1150 pm, 1200 pm, 1250 pm, 1300 pm, 1350 pm, 1400 pm, 1450 pm, or 1500 pm. The length of the microelectrodes typically refers to the longest dimension of the microelectrode(s) measured from the tip, which penetrates a tissue.

[0077] The microelectrodes of the array can be spaced in the array in a manner effective to penetrate into the target tissues (e.g., through the stratum comeum) and to achieve electroporation of cells with the electrical pulses generated by the piezoelectric pulse generator. The microelectrodes are mechanically robust to prevent shearing off, or otherwise damaging, the microelectrodes during insertion or withdrawal in a tissue. The microelectrodes may include a straight or tapered body, which may be cylindrical or square-shaped, and may include a tapered tip portion. The microelectrodes may be manufactured using any suitable method known in the art, for example, lithographic etching technology, photochemical etching, electroplating, wet or dry etching, 3D printing, micro-molding, and laser cutting. The microelectrodes may be solid, have a hollow bore extending from a base to the tip or may have one or more grooves in the sides of the microelectrodes. Method of making the microelectrodes, as well as other components of the MEA, such as the base, and hole for receiving at least one needle, are known to those skilled in the art. For example, molding, 3-D printing, and other suitable techniques can be used to form the base having a hole separately from the microelectrode array, where these parts can be combined to form the MEA, as shown in Figure 2A. The microelectrodes are typically closely spaced to achieve a high field strength in conjunction with microsecond pulses for electroporation of cells in biological tissues. Because the piezoelectric pulses are of microseconds duration, effective electroporation benefits from a field strength >500 V / cm. To achieve such a high field strength, the microelectrodes of the array should have a close spacing, for example, of less than 1 mm apart, i.e., where spacing refers to the distance between a microelectrode (which acts as a cathode) and its nearest neighboring microelectrode (which acts as an anode), and vice versa. This close spacing allows piezoelectric pulses of hundreds of volts to achieve the required field strengths. In some cases, the microelectrodes of the array of the device have a spacing in the array between about 0.1 mm and 10 mm, between 0.2 mm and 5 mm, and between 0.3 mm and 2 mm. In some cases, the spacing is in the range of about 0.5 mm to 1.5 mm. In still other instances, the biocompatible and conductive microelectrodes of the array have: a spacing in the array between about 0.1 mm and 3 mm, between about 0.2 mm and 2.5 mm, between about 0.3 mm and 2 mm, between about 0.5 mm and 1.5 mm, between about 0.5 mm and 1.0 mm, between about 0.5 mm and 0.9 mm, between about 0.5 mm and 0.8 mm, between about 0.5 mm and 0.7 mm, or between about 0.5 mm and 0.6 mm, as well as sub-ranges or individual values contained within; and / or the microelectrodes of the array are arranged in a rectangular, square, triangular, or circular orientation or pattern. Other orientations and patterns are possible. In some instances, the microelectrodes may be in alternating rows, or may be in a checkerboard pattern, or may be in a concentric circle pattern, or may be arranged in other suitable patterns. Rows or columns of microelectrodes can function as positive and negative electrodes.

[0078] In some instances, electroporation may require electric fields on the order of 103V / cm, with shorter pulses requiring larger field strengths. This means that more closely spaced microelectrodes can electroporate with lower voltages. Close electrode spacing can also decrease the electric field penetration depth into the tissue, which can facilitate epidermal targeting and reduce nerve stimulation when used on skin. The pulse length (or decay constant) can be in the order of microseconds (e.g., 1 - 1000 microseconds), and possibly nanoseconds e.g., 1 - 1000 nanoseconds), depending on the piezoelectric crystal utilized. The pulse length can be manipulated, for example, through choosing different dopants utilized in the piezoelectric crystal’s manufacturing process, choosing different dopants, different piezoelectric materials, and other methods known in the art. The pulse length may be in the range of about 1 ns to 1 ms or in the range of about 1 to 100 ps. a. Needle Opening and Needle Component

[0079] As shown in Figure 2A, the MEA includes at least one opening / hole which can receive at least a needle component 110, such as shown in Figure 2C. The needle component has a needle tip. In some instances, the needle component is a hypodermic needle, a hollow microneedle, an insulin syringe needle, or a pen needle. Other needles known in the art may be used. In some instances, the microneedle can be coated and / or dissolvable, as detailed above.

[0080] As shown in Figures 3A and 3B, the needle component 110 is placed into the base 230 through the opening / hole such that the needle tip is exposed adjacent to the microelectrodes of the array 210. In some instances, the MEA may include two, three, four, or more needle components. ii. Piezoelectric Generator and Switch

[0081] The device includes a piezoelectric pulse generator 300, as shown in Figure 4, having a switch 310. The piezoelectric generator includes a piezoelectric crystal which can be selected, without limitation, from lead zirconate titanate (PZT), silicon nitride, barium titanate, quartz, zinc oxide, sodium tungstate, sodium potassium tartrate, tourmaline, lithium niobate, gallium arsenide, aluminum nitride, and combinations thereof. The piezoelectric crystal is a material which exhibits a piezoelectric effect of large magnitude, that is the production of an electric field and thereby a large voltage output with a short time constant in the form of a pulse. This may occur through a mechanical force, pressure, or compression exerted against the crystal’s surface, creating a temporary deformation resulting in the formation of electric charges which are then released as an electrical pulse. Piezoelectric pulse generators are known to those skilled in the art. Suitable piezoelectric pulse generator can be selected for use in the devices which provide the desired performance properties, as detailed below. These generators may be obtained from commercial sources. In addition, methods for their manufacture are also known to those skilled in the art.

[0082] The piezoelectric pulse generator is coupled to a switch, as shown in Figure 4, for activating the piezoelectric pulse generator and causing the generation of one or more electrical pulses. The term “switch” refers to a non-electrical switch, which is a mechanical device or mechanism used to control and activate the generation of electrical currents, and pulses thereof, by the piezoelectric pulse generator. A non-electrical switch is preferred for the device as it does not require the use of electricity or electronic components therein.

[0083] In some instances, the switch triggers a spring-latch hammer mechanism configured to strike a surface of the piezoelectric crystal of the piezoelectric pulse generator. In some instances, there is an optional pin (such as a metal pin) disposed between the spring-latch hammer mechanism and the piezoelectric crystal.

[0084] In some instances, the switch is a toggle switch with a latch configured to release a hammer, configured to strike a surface of the piezoelectric crystal of the piezoelectric pulse generator effectively, such as driven by decompression of a spring. In some instances, an optional pin (such as a metal pin) is disposed between the latch and the piezoelectric crystal. In some instances, the latch can be a wedge controlling latch, where the wedge acts to unlock the hammer (pushes it out of a lock), which results in a spring decompressing the hammer and launching it at the piezoelectric crystal to produce an electric force.

[0085] In some instances, the piezoelectric pulse generator includes a casing for the piezoelectric crystal and electrical connections inclusive of a first electrode and a second electrode which extend from the casing, where the first and second electrodes provide electrical contact points where the MEA can be connected or mated to in order to receive the electrical pulse(s) generated by the piezoelectric pulse generator.

[0086] The array of biocompatible and conductive microelectrodes is electrically coupled to the piezoelectric pulse generator. The piezoelectric pulse generator may include any mechanism effective to generate a suitable electrical pulse from a piezoelectric crystal. In some instances, the piezoelectric pulse generator includes (i) a piezoelectric crystal, (ii) a spring-latch hammer mechanism configured to strike a surface of the piezoelectric crystal effective to generate the electrical pulse, and (iii) electrical connections for conducting the electrical pulse to the microelectrodes of the array.

[0087] In certain instances, the piezoelectric pulse generator, when activated by the switch, generates one or more electrical pulses having a peak voltage absolute value of between about 10 V and 35,000 V, 10 V and 30,000 V, 10 V and 20,000 V, 10 V and 10,000 V, 20,000 V and 30,000 V, between about 50 V and 5,000 V, between about 100 V and 1,000 V, or between about 200 V and 500 V when measured in air, or sub-ranges or individual values contained within the aforementioned ranges. In some instances, the piezoelectric pulse generator, when activated by the switch, generates one or more electrical pulses having a peak voltage absolute value of between about 20,000 V and 30,000 V or 23,000 V and 28,000 V, when measured in air, or sub-ranges or individual values contained within the aforementioned ranges. In some instances, the one or more electrical pulses have a peak voltage absolute value of between about 100 V and 1000 V, about 100 V and 900 V, about 100 V and 800 V, about 100 V and 700 V, about 100 V and 600 V, about 100 V and 500 V, about 100 V and 400 V, about 100 V and 300 V, about 100 V and 200 V, about 200 V and 400 V, between about 200 V and 350 V, between about 200 V and 300 V, or between about 200 V and 250 V when measured in a tissue, or subranges or individual values contained within the aforementioned ranges.

[0088] In some instances, the one or more electrical pulses have a ratio of absolute value of peak voltage to absolute value of peak-to-peak voltage between about 0.1 and 10, between about 0.3 and 5, or between about 0.5 and 2, or sub-ranges or individual values contained within the aforementioned ranges. In certain instances, the one or more electrical pulses have a peak current absolute value between about 0.001 A and 1,000 A, between about 0.01 A and 500 A, between about 0.1 A and 100 A, or between about 1 A and 50 A when measured in a tissue, or sub-ranges or individual values contained within the aforementioned ranges. In some instances, the one or more electrical pulses have a peak static voltage absolute value of between about 100

[0089] V and 35,000 V, between about 1,000 V and 30,000 V, or between about 15,000 V and 35,000

[0090] V when measured in air, or sub-ranges or individual values contained within the aforementioned ranges. In still other instances, the one or more electrical pulses can produce an electric field strength of between about 100 V / cm and 30,000 V / cm, between about 200 V / cm and 10,000 V / cm, between about 300 V / cm and 5,000 V / cm, or between about 500 V / cm and 3,500 V / cm, or sub-ranges or individual values contained within the aforementioned ranges. In yet other instances, the one or more electrical pulses generated can have an initial pulse length of between about 1 ps and 10,000 ps, between about 1 ps and 1,000 ps, between about 1 ps and 100 ps, between about 3 ps and 100 ps , between about 5 ps and 50 ps , or between about 10 ps and 30 ps, or sub-ranges or individual values contained within the aforementioned ranges. In some instances, the one or more electrical pulses generated can have a ratio of initial pulse length to total pulse length between about 1.5 and 100, between about 2 and 50, or between about 3 and 20. Total pulse length should be greater than initial pulse length.

[0091] The device may include a casing surrounding the piezoelectric crystal, which includes electrical connections for relaying the pulse to the microelectrodes of the array. The casing may include a lower electrode and a side electrode, which extends from the casing.

[0092] In some instances, the device may operate as follows: After the microelectrodes of the array are inserted into a tissue, such as skin, mucosa, or other biological tissue, a user exerts a force against the switch, such as a thumb-toggle switch, which compresses a lower spring, and pushes a wedge towards the hammer locked in a latch. When the user pushes all the way, the wedge forces the hammer out of the latch which subsequently strikes a pin sitting against the piezoelectric crystal to concentrate the force. The voltage output generated by the piezoelectric pulse generator may then be directed to the microelectrodes of the array of the device. An upper spring may then be decompressed to reset the hammer and latch into the original locked state. The user may repeat the operation to generate additional electrical pulses, as needed to provide an effective electroporation of the tissue(s) for delivery of the agent(s) into the tissue and cells thereof.

[0093] The electroporation portion of the devices described herein provides an electrical pulse capable of increasing deliver}' of agents, such as drugs and molecules into the tissue and cells therein. The use of piezoelectric crystals as the source of the electric pulses induces membrane permeabilization through a high-voltage, short time-constant pulse (e.g., microseconds), which is believed to induce a temporary change in the cell membranes of cells in the electric field produced by the microelectrodes of the array during an electric pulse(s), which includes cells in contact with, or in the vicinity of the microelectrodes, allowing agent(s) to enter the tissue cells, to produce an intended effect.

[0094] In some instances, the piezoelectric pulse generator produces bipolar, oscillatory' pulses, which may electroporate cells more effectively compared to conventional monopolar or exponential-decay or square- wave pulses.

[0095] The foregoing pulse values refer to pulses in biological tissues, such as the skin, unless they are identified as static voltages (i.e., peak static voltage, peak-to-peak static voltage). It is noted that similar, conventional piezoelectric devices are designed to make sparks in air. In contrast, the piezoelectric pulse generators described herein are coupled with microelectrodes of an array which are used to pass current through a conductive medium (i.e., no sparks). In certain instances, a bipolar oscillating pulse is used for electroporation instead of a monopolar pulse, often in the form of an exponential-decay or square wave pulse, which is more conventionally used for electroporation. This bipolar oscillating pulse may be a natural result of the compression and extension of piezoelectric crystals induced by a spring shock in the case containing the piezoelectric crystal. Compared to conventional monopolar pulses, bipolar oscillating pulses are not only able to produce a dielectric breakdown of the cell membrane but can also produce a sonicating motion in the cell membrane, inducing more effective cell poration. Further, oscillating pulses may provide better cell viability by avoiding polarizing the cell membrane beyond the critical potential for an extensive period, therefore, preventing irreversible rupture of the cell membrane. While for non-oscillatory pulses, the initial and total pulse lengths are the same, the pulses of the devices described herein may be oscillatory, such that the initial and total pulse lengths may not be the same. Moreover, while conventional electroporators generate substantially monopolar pulses, the devices and methods described herein may use bipolar pulses, wherein each pulse alternates between positive and negative. iii. Syringe Component

[0096] In some instances, the devices described herein may include a syringe component. As shown in Figure 5A, the syringe may include a container 126, which can hold one or more agents, a plunger 125 which can be used to administer the agents when actuated, and a delivery end which optionally includes an attachment or lock, such as a Luer lock, used for connecting and attaching a needle component. For example, Figure 5B shows a non-limiting illustration of a syringe coupled to a needle component 110 and an MEA 200, where such an assembled unit may be placed directly into device casing 130. In some instances, the assembled unit can optionally be held in the device casing by pressure (clicks into the device casing) and / or may be held by optional screws or pins.

[0097] In some other instances, the syringe component need not rely on a plunger, which is manually pressed to cause delivery of an agent(s) contained within the syringe but can rely instead on, for example, air pressure to cause delivery of the agent(s) from the syringe container. Such pressure-based systems (z.e., jet injectors) are known to those skilled in the art and can be used in the device in lieu of the common plunger-style syringe illustrated. iv. Other Device Components

[0098] As noted above, the device includes a device casing, as shown in Figures 1 A-1C. The device casing may have any suitable shape and can be made from any suitable materials. The casing may be manufactured using known methods, such as casting, molding, 3-D printing, and may be made of plastic. The casing may include a finger rest. The device casing can receive and hold the other device components including the piezoelectric pulse generator and switch, as well as the syringe, needle component, and MEA.

[0099] III. Uses of Devices for Delivering Agents

[0100] The devices described above can be used for various medical applications. In one nonlimiting instance, the device may be used in a method of delivering an agent into or across a biological tissue, the method including the steps of:

[0101] (a) positioning a device adjacent to a biological tissue site, the device including: a piezoelectric pulse generator; an array of biocompatible and conductive microelectrodes electrically coupled to the piezoelectric pulse generator; a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses; a syringe comprising a container holding at least one agent; wherein the array of biocompatible and conductive microelectrodes includes at least one needle for delivery of the at least one agent embedded therein; wherein the syringe is coupled to the at least one needle for administering the at least one agent into a biological tissue by actuation of the syringe;

[0102] (b) contacting the array of biocompatible and conductive microelectrodes including the at least one needle embedded therein into the target biological tissue site;

[0103] (c) activating the switch to generate and deliver one or more electrical pulses through the array of biocompatible and conductive microelectrodes into the target biological tissue site to electroporate cells at the target biological tissue site; and

[0104] (d) administering the at least one agent into the target biological tissue site through the at least one needle by actuation of the syringe.

[0105] In some instances of the method, step (c) and step (d) are performed concurrently. In some instances, step (d) is performed following step (c). In other instances, step (c) is performed following step (d). In still other instances, step (c) can be repeated at least once or two or more times.

[0106] In some instances, contacting during step (b) refers to insertion and / or penetration of the microelectrodes of the array into the target biological tissue.

[0107] Typically, the methods include manually applying a force to the device effective to penetrate a biological tissue site surface with the array of biocompatible and conductive microelectrodes, and then manually pressing the switch on the device that triggers a mechanical force onto a piezoelectric pulse generator to produce one or more electric pulses effective to induce electroporation (z.e., pore formation) in the cells of the biological tissue site. Depressing the plunger of the syringe can actuate agent release, from the syringe container.

[0108] The methods described herein are used to administer agents, typically a drug, to a subject’s biological tissue site with the aid of electroporation.

[0109] In some instances of the method, the target biological tissue site includes mammalian skin. In some instances, the target biological tissue site is mammalian skin dermis. In still other instances, the target biological tissue site is mammalian skin epidermis. In certain instances, the target biological tissue site is or includes a mucosal membrane. A mucosal membrane, also known as a mucous membrane, is a type of epithelial tissue that lines various cavities and structures within the body that are exposed to the external environment.

[0110] In some instances of the method, the one or more electrical pulses have a peak voltage absolute value between about 100 V and 1000 V, a peak current absolute value between about 0.001 A and 50 A, a peak static voltage absolute value between about 15,000 V and 35,000 V, an initial pulse length of between about 1 ps and 100 ps, or a combination thereof. Other parameters of the device are possible as detailed in device Section II.

[0111] The methods described can be used to deliver agents which are large molecule drugs into body tissues of a subject using electroporation without the use of batteries, capacitors, or other conventional electric power storage devices, and without the use of electricity from an external source (e.g., not plugged into an electrical outlet).

[0112] In some instances, the electric field is localized to the target biological tissue site, such as the skin and especially to the epidermis, where localization to the epidermis means that the majority of cells experiencing electroporation conditions are located in the epidermis and not in the dermis. This localization is facilitated by the combination of limiting penetration of the electric field to deeper tissues and by the fact that cell density in viable epidermis is much greater than in dermis. Targeting the electric field and resulting electroporation to the skin, for example, and especially the epidermis improves immunogenicity and allows for reduced side effects. Unlike dermis, which is largely acellular, the epidermis is densely populated with cells, including keratinocytes as well as potent antigen-presenting cells, such as dendritic cells, including Langerhans cells. Targeting certain agents, such as an antigen, to these epidermal cells has been shown to improve immune responses compared to intramuscular injection. Nevertheless, diffusion of antigens produced in epidermal cells into the upper dermis can also be beneficial, due to the presence of dermal dendritic cells and a rich vasculature that enables drainage to lymph nodes, which also increases immunogenicity.

[0113] Moreover, localizing the electric field to the epidermis can reduce nerve stimulation, thereby making electroporation more tolerable. Of particular concern is stimulation of motor nerves and muscle cells below the skin, which can cause violent twitching reported for skin or muscle electroporation in other contexts. Such contractions are not expected when electroporating with the MEAs described herein which can localize the electric field superficially, far away from muscles.

[0114] In most instances, the present microelectrodes of the array can be used for controlled and selective penetration into the epidermis, and possibly a portion of the dermis, as needed. In most instances, the microelectrodes would not fully cross the dermis and would not contact tissues below the dermis. Because the electric field produced when pulsing these microelectrodes is strongest in the biological tissue between oppositely charged electrodes, tissue deeper than the penetration depth of the microelectrodes receives weaker electric fields that is less likely to cause electroporation. A. Agents for Delivery

[0115] The devices and methods described can be used to deliver at least one agent into or across a biological tissue. In some instances, more than one type of agent can be delivered using the device and methods described.

[0116] The devices and methods described can be used to deliver agents known to those skilled in the art. In some instances, the at least one agent can be selected from a nucleic acid (such as DNA and / or RNA, nucleic acid encoding a vaccine, nucleic acid encoding a therapeutic protein, nucleic acid encoding a hormone, or a nucleic acid encoding a monoclonal antibody), a DNA- plasmid, a minicircle DNA plasmid, an mRNA, a self- amplifying RNA, a circular RNA, a transfer RNA, a chemotherapeutic agent, a biologic, a prophylactic, a DNA-launched selfamplifying RNA, a DNA-launched virus, a linear DNA, and combinations thereof. In some instances, the at least one agent is a therapeutic wherein the therapeutic is a RNA therapeutic or DNA therapeutic.

[0117] In yet other instances, the at least one agent can be selected from a wide variety of drugs, including small molecules, proteins, nucleic acids-based compounds, and biologies. Small molecules generally refers to therapeutic, prophylactic, or diagnostic agents known in the art, which have a molecular weight of less than about 500 Daltons. The range of drugs includes biologies, which may be include of antigens (substances derived from or mimicking foreign substances to induce an immune response when introduced) synthetically synthesized in the form of nucleic acids or proteins or naturally obtained from relevant sources; nucleic acids such as DNA, RNA, or other polynucleotide compounds in plasmid or linear form; peptides (amino acid sequences); viral vectors or viruses; vaccines of any type; gene therapies such as CAR-T cell therapy to treat cancers; immunotherapies; chemically or biologically synthesized proteins; and / or any combination of these.

[0118] In still other instances, the at least one agent can be a vaccine, wherein the vaccine can be an RNA vaccine or a DNA vaccine.

[0119] In yet other instances, the at least one agent can be a therapeutic, wherein the therapeutic can be an RNA therapeutic or a DNA therapeutic.

[0120] In some instances, the methods use the microelectrodes to target delivery to the tissue, such as skin, which can provide greater immunogenicity for DNA, RNA, and other vaccines compared to vaccines delivered into muscle.

[0121] In still other instances, the devices and methods described can be used to administer any suitable agent, particularly one where electroporation can facilitate cellular uptake of the agent.

[0122] The agent may be essentially any therapeutic or prophylactic agent known in the art or developed. In cases, the agent is a small drug molecule, a biologic, or a vaccine. Non-limiting examples include therapeutic proteins (such as antibodies, enzymes, growth factors, hormones, interferons, interleukins, engineered proteins, and vaccines), RNA (such as messenger RNA (mRNA), self- amplifying RNA (saRNA), circular RNA (circRNA), RNA interference (RNAi) including short interfering RNA (siRNA) and micro RNA (miRNA), antisense RNA (asRNA) or short hairpin RNA (shRNA)or RNA aptamers), DNA (such as plasmids, oligonucleotides, DNA aptamers, DNAzymes), anti-cancer drugs, antibacterial drugs, inhibitors (of, for example, protein synthesis, cell wall synthesis, enzymatic activity, biochemical pathways), drugs affecting intracellular or intercellular signaling, drugs affecting gene regulation. In one instance, the agent is a vaccine, where the vaccine may be selected to be effective against any of a variety of viruses, bacteria, or other pathogens, including but not limited to SARS-CoV-2, Ebola, influenza, etc. The vaccine may be formed of mRNA and / or DNA encoding one or more antigens, or may be a vaccine effective against multiple variants of a disease.

[0123] IV. Kits

[0124] The devices described are handheld devices which can be for single-use or repeated use. The devices described above can be included in a kit. In some instances, the kit can include the device including at least the device casing, the piezoelectric pulse generator, the switch, and the MEA, each of which is as described above. In some instances, the kit may further include at least a syringe and a needle component. In yet other instances, the kit may also include one or more agents. A kit may also include instructions for use. In some instances, a kit may also include packs of microelectrode array cartridges. In still other instances, the kit may also include blister packs of at least one agent and multiple reusable tips for administration of the at least one agent.

[0125] The disclosed compositions and methods can be further understood through the following numbered paragraphs.

[0126] Paragraph 1. A microelectrode array comprising: an array of biocompatible and conductive microelectrodes comprising an opening for at least one needle therein; wherein a syringe comprising a container for holding at least one agent can be coupled to the at least one needle for delivery of the at least one agent.

[0127] Paragraph 2. The microelectrode array of paragraph 1 , further comprising a base from which the array of biocompatible and conductive microelectrodes extends.

[0128] Paragraph 3. The microelectrode array of any one of paragraphs 1-2, wherein the biocompatible and conductive electrodes are formed of a metal; or wherein the biocompatible and conductive electrodes are formed of a mixture of epoxy and graphite, a mixture of glass and platinum, or a conductive ceramic.

[0129] Paragraph 4. The microelectrode array of paragraph 3, wherein the metal is stainless steel.

[0130] Paragraph 5. The microelectrode array of any one of paragraphs 1-4, wherein the biocompatible and conductive microelectrodes of the array extend from one or more metal plates.

[0131] Paragraph 6. The microelectrode array of paragraph 5, wherein a linear array of the biocompatible and conductive microelectrodes extends from one edge of each of the one or more metal plates.

[0132] Paragraph 7. The microelectrode array of paragraph 5 or 6, wherein the one or more plates are parallel to each other and spaced apart from one another.

[0133] Paragraph 8. The microelectrode array of any one of paragraphs 1-4, wherein the biocompatible and conductive microelectrodes of the array extend from a single metal plate, and the array is a two-dimensional array.

[0134] Paragraph 9. The microelectrode array of any one of paragraphs 1-4, wherein the biocompatible and conductive microelectrodes of the array extend from one or more metal plates and the array is a two-dimensional array.

[0135] Paragraph 10. The microelectrode array of any one of paragraphs 1-4, wherein the biocompatible and conductive microelectrodes of the array extend from at least one non- electrically conductive plate and wherein electrical connections are provided between the biocompatible and conductive microelectrodes.

[0136] Paragraph 11. The microelectrode array of paragraph 10, wherein the electrical connections are located on a surface of the at least one non-electrically conductive plate.

[0137] Paragraph 12. The microelectrode array of paragraph 10 or 11, wherein the electrical connections cross from a first side of the at least one non-electrically conductive plate to an opposed second side of the plate through holes in the at least one non-electrically conductive plate.

[0138] Paragraph 13. The microelectrode array of any one of paragraphs 1-12, wherein the biocompatible and conductive microelectrodes of the array have an area between about 1 mm2and 50 cm2or about 1 mm2and 25 cm2.

[0139] Paragraph 14. The microelectrode array of any one of paragraphs 1-13, wherein the array of biocompatible and conductive microelectrodes comprises from: (1) about 2 to 3000 microelectrodes, about 2 to 2000 microelectrodes, or about 2 to 1000 microelectrodes; or (2) at least: about 25 microelectrodes, about 50 microelectrodes, about 100 microelectrodes, about 150 microelectrodes, about 200 microelectrodes, about 250 microelectrodes, about 300 microelectrodes, about 350 microelectrodes, about 400 microelectrodes, about 450 microelectrodes, about 500 microelectrodes, about 550 microelectrodes, about 600 microelectrodes, about 650 microelectrodes, about 700 microelectrodes, about 750 microelectrodes, about 800 microelectrodes, about 850 microelectrodes, about 900 microelectrodes, about 950 microelectrodes, about 1000 microelectrodes, about 1500 microelectrodes, about 2000 microelectrodes, about 2500 microelectrodes, or about 3000 microelectrodes.

[0140] Paragraph 15. The microelectrode array of any one of paragraphs 1-14, wherein the biocompatible and conductive microelectrodes of the array each independently have a length from 10 pm to 8 mm; or wherein the biocompatible and conductive microelectrodes of the array each independently have a length of at least about 500 pm, 600 pm, 650 pm, 700 pm, 750 pm, 800 pm, 900 pm, 950 pm, 1000 pm, 1050 pm, 1100 pm, 1150 pm, 1200 pm, 1250 pm, 1300 pm, 1350 pm, 1400 pm, 1450 pm, or 1500 pm.

[0141] Paragraph 16. The microelectrode array of any one of paragraphs 1-15, wherein the biocompatible and conductive microelectrodes of the array have: a spacing in the array between about 0.1 mm and 3 mm, between about 0.2 mm and 2.5 mm, between about 0.3 mm and 2 mm, between about 0.5 mm and 1.5 mm, between about 0.5 mm and 1.0 mm, between about 0.5 mm and 0.9 mm, between about 0.5 mm and 0.8 mm, between about 0.5 mm and 0.7 mm, or between about 0.5 mm and 0.6 mm; and / or the microelectrodes of the array are arranged in a rectangular, square, triangular, or circular orientation.

[0142] Paragraph 17. The microelectrode array of any one of paragraphs 1-16, wherein the at least one needle is a hypodermic needle, a hollow microneedle, an insulin syringe needle, or a pen needle.

[0143] Paragraph 18. The microelectrode array of any one of paragraphs 1-17, wherein the microelectrode array further comprises a patch of microneedles which are optionally coated and / or dissolvable.

[0144] Paragraph 19. A device comprising: a piezoelectric pulse generator; an array of biocompatible and conductive microelectrodes electrically coupled to the piezoelectric pulse generator; and a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses; wherein the array of biocompatible and conductive microelectrodes comprises an opening for insertion of at least one needle therein for delivery of at least one agent; wherein a syringe comprising a container for holding the at least one agent can be coupled to the at least one needle and the at least one agent can be administered into a biological tissue by actuation of the syringe; wherein insertion of the array of biocompatible and conductive microelectrodes into the biological tissue and activation of the piezoelectric pulse generator generates the one or more electrical pulses through the array of biocompatible and conductive microelectrodes to electroporate cells in the biological tissue to permit administration of the at least one agent into the electroporated cells.

[0145] Paragraph 20. The device of paragraph 19, the device further comprising at least one needle.

[0146] Paragraph 21. The device of paragraph 20, wherein the at least one needle is a hypodermic needle, a hollow microneedle, an insulin syringe needle, or a pen needle.

[0147] Paragraph 22. The device of any one of paragraphs 19-21, the device further comprising a syringe.

[0148] Paragraph 23. The device of any one of paragraphs 19-22, further comprising (i) a base from which the array of biocompatible and conductive microelectrodes extends, and (ii) a housing which contains the piezoelectric pulse generator and the switch.

[0149] Paragraph 24. The device of any one of paragraphs 19-23, further comprising (i) a base from which the array of biocompatible and conductive microelectrodes extends, and (ii) a housing which contains the base, the piezoelectric pulse generator, and the switch.

[0150] Paragraph 25. The device of any one of paragraphs 19-24, the device further comprising the at least one agent.

[0151] Paragraph 26. The device of paragraph 25, wherein the at least one agent is selected from the group consisting of a nucleic acid (such as DNA and / or RNA, nucleic acid encoding a vaccine, nucleic acid encoding a therapeutic protein, nucleic acid encoding a hormone, or a nucleic acid encoding a monoclonal antibody), a DNA-plasmid, a minicircle DNA plasmid, an mRNA, a self- amplifying RNA, a circular RNA, a transfer RNA, a chemotherapeutic agent, a biologic, a prophylactic, a DNA-launched self-amplifying RNA, a DNA-launched virus, a linear DNA, and combinations thereof. Paragraph 27. The device of paragraph 24, wherein the at least one agent is a therapeutic.

[0152] Paragraph 28. The device of paragraph 27, wherein the vaccine is an RNA therapeutic or a DNA therapeutic.

[0153] Paragraph 29. The device of any one of paragraphs 19-28, wherein the biocompatible and conductive microelectrodes are formed of a metal; or wherein the biocompatible and conductive electrodes are formed of a mixture of epoxy and graphite, a mixture of glass and platinum, or a conductive ceramic.

[0154] Paragraph 30. The device of paragraph 29, wherein the metal is stainless steel.

[0155] Paragraph 31. The device of any one of paragraphs 19-30, wherein the biocompatible and conductive microelectrodes of the array extend from one or more metal plates which are configured to conduct the one or more electrical pulses from the piezoelectric pulse generator to the microelectrodes of the array.

[0156] Paragraph 32. The device of paragraph 31 , wherein a linear array of the biocompatible and conductive microelectrodes extends from one edge of each of the one or more metal plates.

[0157] Paragraph 33. The device of paragraph 31 or 32, wherein the one or more plates are parallel to each other and spaced apart from one another.

[0158] Paragraph 34. The device of any one of paragraphs 19-30, wherein the biocompatible and conductive microelectrodes of the array extend from a single metal plate, and the array is a two-dimensional array.

[0159] Paragraph 35. The device of any one of paragraphs 19-30, wherein the biocompatible and conductive microelectrodes of the array extend from one or more metal plates and the array is a two-dimensional array.

[0160] Paragraph 36. The device of any one of paragraphs 19-30, wherein the biocompatible and conductive microelectrodes of the array extend from at least one non-electrically conductive plate and wherein electrical connections are provided between the biocompatible and conductive microelectrodes and configured to conduct the one or more electrical pulses from the piezoelectric pulse generator to the microelectrodes of the array.

[0161] Paragraph 37. The device of paragraph 36, wherein the electrical connections are located on a surface of the at least one non-electrically conductive plate.

[0162] Paragraph 38. The device of paragraph 36 or 37, wherein the electrical connections cross from a first side of the at least one non-electrically conductive plate to an opposed second side of the plate through holes in the at least one non-electrically conductive plate. Paragraph 39. The device of any one of paragraphs 19-38, wherein the piezoelectric pulse generator comprises a piezoelectric crystal selected from the group consisting of lead zirconate titanate (PZT), silicon nitride, barium titanate, quartz, zinc oxide, sodium tungstate, sodium potassium tartrate, tourmaline, lithium niobate, gallium arsenide, aluminum nitride, and combinations thereof.

[0163] Paragraph 40. The device of any one of paragraphs 19-39, wherein the switch triggers a spring-latch hammer mechanism configured to strike a surface of the piezoelectric crystal of the piezoelectric pulse generator.

[0164] Paragraph 41. The device of paragraph 40, further comprising a pin disposed between the spring-latch hammer mechanism and the piezoelectric crystal.

[0165] Paragraph 42. The device of any one of paragraphs 19-39, wherein the switch is a toggle switch with a latch configured to release a hammer, configured to strike a surface of the piezoelectric crystal of the piezoelectric pulse generator effective.

[0166] Paragraph 43. The device of paragraph 42, further comprising a pin disposed between the latch and the piezoelectric crystal.

[0167] Paragraph 44. The device of any one of paragraphs 19-43, further comprising a casing for the piezoelectric crystal; and wherein electrical connections comprised of a first electrode and a second electrode extend from the casing.

[0168] Paragraph 45. The device of paragraph 44, further comprising a cartridge that contains the array of biocompatible and conductive microelectrodes and comprises a first receptacle for mating engagement with the first electrode and a second receptacle for mating engagement with the second electrode, the first and second receptacles are in electrical communication with the microelectrodes of the array.

[0169] Paragraph 46. The device of any one of paragraphs 19-45, wherein the microelectrodes of the array are arranged to be inserted into the biological tissue having an area between about 1 mm2and 50 cm2or about 1 mm2and 25 cm2.

[0170] Paragraph 47. The device of any one of paragraphs 19-46, wherein the array of biocompatible and conductive microelectrodes comprises from: (1) about 2 to 3000 microelectrodes, about 2 to 2000 microelectrodes, or about 2 to 1000 microelectrodes; or (2) at least: about 25 microelectrodes, about 50 microelectrodes, about 100 microelectrodes, about 150 microelectrodes, about 200 microelectrodes, about 250 microelectrodes, about 300 microelectrodes, about 350 microelectrodes, about 400 microelectrodes, about 450 microelectrodes, about 500 microelectrodes, about 550 microelectrodes, about 600 microelectrodes, about 650 microelectrodes, about 700 microelectrodes, about 750 microelectrodes, about 800 microelectrodes, about 850 microelectrodes, about 900 microelectrodes, about 950 microelectrodes, about 1000 microelectrodes, about 1500 microelectrodes, about 2000 microelectrodes, about 2500 microelectrodes, or about 3000 microelectrodes.

[0171] Paragraph 48. The device of any one of paragraphs 19-47, wherein the microelectrodes of the array each independently have a length from 10 pm to 8 mm; or wherein the biocompatible and conductive microelectrodes of the array each independently have a length of at least about 500 pm, 600 pm, 650 pm, 700 pm, 750 pm, 800 pm, 900 pm, 950 pm, 1000 pm, 1050 pm, 1100 pm, 1150 pm, 1200 pm, 1250 pm, 1300 pm, 1350 pm, 1400 pm, 1450 pm, or 1500 pm.

[0172] Paragraph 49. The device of any one of paragraphs 19-48, wherein the array of biocompatible and conductive microelectrodes is replaceable; and / or wherein the piezoelectric pulse generator and the switch are each independently single-use or are reusable.

[0173] Paragraph 50. The device of any one of paragraphs 19-49, wherein the one or more electrical pulses have a peak voltage absolute value of between about 10 V and 35,000 V, 10 V and 35,000 V, 10 V and 30,000 V, 10 V and 20,000 V, 10 V and 10,000 V, 20,000 V and 30,000 V, between about 50 V and 5,000 V, between about 100 V and 1,000 V, or between about 200 V and 500 V when measured in air; or wherein the one or more electrical pulses have a peak voltage absolute value of between about 100 V and 1000 V, about 100 V and 900 V, about 100 V and 800 V, about 100 V and 700 V, about 100 V and 600 V, about 100 V and 500 V, about 100 V and 400 V, about 100 V and 300 V, about 100 V and 200 V, about 200 V and 400 V, between about 200 V and 350 V, between about 200 V and 300 V, or between about 200 V and 250 V when measured in a tissue.

[0174] Paragraph 51. The device of any one of paragraphs 19-50, wherein the one or more electrical pulses have a ratio of absolute value of peak voltage to absolute value of peak-to-peak voltage between about 0.1 and 10, between about 0.3 and 5, or between about 0.5 and 2.

[0175] Paragraph 52. The device of any one of paragraphs 19-51, wherein the one or more electrical pulses have a peak current absolute value between about 0.001 A and 1,000 A, between about 0.01 A and 500 A, between about 0.1 A and 100 A, or between about 1 A and 50 A.

[0176] Paragraph 53. The device of any one of paragraphs 19-52, wherein the one or more electrical pulses have a peak static voltage absolute value of between about 100 V and 35,000 V, between about 1,000 V and 30,000 V, or between about 15,000 V and 35,000 V when measured in air. Paragraph 54. The device of any one of paragraphs 19-53, wherein the one or more electrical pulses produce an electric field strength of between about 100 V / cm and 30,000 V / cm, between about 200 V / cm and 10,000 V / cm, between about 300 V / cm and 5,000 V / cm, or between about 500 V / cm and 3,500 V / cm.

[0177] Paragraph 55. The device of any one of paragraphs 19-54, wherein the one or more electrical pulses generated have an initial pulse length of between about 1 ps and 10,000 ps, between about I s and 1,000 ps, between about 1 ps and 100 ps, between about 3 ps and 100 ps , between about 5 ps and 50 ps , or between about 10 ps and 30 ps.

[0178] Paragraph 56. The device of any one of paragraphs 19-55, wherein the one or more electrical pulses generated have a ratio of initial pulse length to total pulse length between about 1.5 and 100, between about 2 and 50, or between about 3 and 20.

[0179] Paragraph 57. The device of any one of paragraphs 19-56, wherein the microelectrodes of the array have: a spacing in the array between about 0.1 mm and 3 mm, between about 0.2 mm and 2.5 mm, between about 0.3 mm and 2 mm, between about 0.5 mm and 1.5 mm, between about 0.5 mm and 1.0 mm, between about 0.5 mm and 0.9 mm, between about 0.5 mm and 0.8 mm, between about 0.5 mm and 0.7 mm, or between about 0.5 mm and 0.6 mm; and / or the microelectrodes of the array are arranged in a rectangular, square, triangular, or circular orientation.

[0180] Paragraph 58. The device of any one of paragraphs 19-57, wherein the device further comprises a patch or a coating, which are optionally dissolvable

[0181] Paragraph 59. A method of delivering an agent into or across a biological tissue, the method comprising the steps of:

[0182] (a) positioning a device adjacent to a biological tissue site, the device comprising: a piezoelectric pulse generator; an array of biocompatible and conductive microelectrodes electrically coupled to the piezoelectric pulse generator; a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses; a syringe comprising a container holding at least one agent; wherein the array of biocompatible and conductive microelectrodes comprises at least one needle for delivery of the at least one agent embedded therein; wherein the syringe is coupled to the at least one needle for administering the at least one agent into a biological tissue by actuation of the syringe; (b) contacting the array of biocompatible and conductive microelectrodes comprising the at least one needle embedded therein into the target biological tissue site;

[0183] (c) activating the switch to generate and deliver one or more electrical pulses through the array of biocompatible and conductive microelectrodes into the target biological tissue site to electroporate cells at the target biological tissue site; and

[0184] (d) administering the at least one agent into the target biological tissue site through the at least one needle by actuation of the syringe.

[0185] Paragraph 60. The method of paragraph 59, wherein step (c) and step (d) are performed concurrently.

[0186] Paragraph 61. The method of paragraph 59, wherein step (d) is performed following step (c).

[0187] Paragraph 62. The method of paragraph 59, wherein step (c) is performed following step (d).

[0188] Paragraph 63. The method of any one of paragraphs 59-62, wherein step (c) is repeated at least once.

[0189] Paragraph 64. The method of any one of paragraphs 59-63, wherein the target biological tissue site comprises mammalian skin.

[0190] Paragraph 65. The method of any one of paragraphs 59-64, wherein the target biological tissue site is mammalian skin dermis.

[0191] Paragraph 66. The method of any one of paragraphs 59-64, wherein the target biological tissue site is mammalian skin epidermis.

[0192] Paragraph 67. The method of any one of paragraphs 59-64, wherein the target biological tissue site is or comprises a mucosal membrane.

[0193] Paragraph 68. The method of any one of paragraphs 59-67, wherein the one or more electrical pulses have a peak voltage absolute value between about 100 V and 1000 V, a peak current absolute value between about 0.001 A and 50 A, a peak static voltage absolute value between about 15,000 V and 35,000 V, an initial pulse length of between about 1 ps and 100 ps, or a combination thereof.

[0194] Paragraph 69. The method of any one of paragraphs 59-68, wherein the at least one agent is selected from the group consisting of a nucleic acid (such as DNA and / or RNA, nucleic acid encoding a vaccine, nucleic acid encoding a therapeutic protein, nucleic acid encoding a hormone, or a nucleic acid encoding a monoclonal antibody), a DNA-plasmid, a minicircle DNA plasmid, an mRNA, a self- amplifying RNA, a circular RNA, a transfer RNA, a chemotherapeutic agent, a biologic, a prophylactic, a DNA-launched self-amplifying RNA, a DNA-launched virus, a linear DNA, and combinations thereof.

[0195] Examples

[0196] Example 1: Ex Vivo Testing in Porcine Skin Demonstrates Effective VID Injections at Multiple Needle Depths Using Piezopen

[0197] Materials and Methods

[0198] The objective of this study was to evaluate the efficacy and efficiency of variable intradermal deliver}' (VID) injection across different needle lengths using the integrated Piezopen device in an ex vivo porcine skin model. Pig ears (Pel-Freez Biologicals) were used as the tissue model for injection testing.

[0199] VID injections were performed using needle lengths of 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, and 1.5 mm. After each injection, a visual inspection was conducted to confirm the formation of a bleb — a small blister or bubble on the tissue surface resulting from fluid injection. The presence of a distinct, white circular bleb indicated a successful VID injection at the intended depth. Numbers representing the needle length were marked at the top of each bleb, while numbers representing the volume of the injection were marked at the bottom of each bleb on the porcine skin.

[0200] Results

[0201] The results demonstrated success in producing VID injections ex vivo using the integrated Piezopen device. Bleb formation indicated that needle lengths of 1.0 mm, 1.1 mm, and 1.2 mm reliably achieved successful VID injections, confirming delivery within the intended dermal layer (data not shown). In contrast, needle lengths of 1.3 mm, 1.4 mm, and 1.5 mm resulted in subcutaneous (SC) injections, as these did not produce the characteristic bleb associated with intradermal delivery (data not shown). These findings suggest that needle lengths of up to about 1.2 mm are optimal for VID injection using the Piezopen in an ex vivo porcine skin model.

[0202] Example 2: Ex Vivo Testing in Human Skin Demonstrates Effective VID Injections Using Piezopen

[0203] Objective of Study

[0204] The objectives of this study were to: (i) establish a protocol for imaging live human skin samples ex vivo using an in vivo imaging system (IVIS) and determine the optimal needle length for effective vertical intradermal (VID) drug deliver}', (ii) evaluate the impact of electroporation (EP) on expression kinetics in ex vivo human skin samples, and (iii) benchmark the performance and effectiveness of the Piezopen device against the current state-of-the-art electroporator, BTX.

[0205] Materials and Methods

[0206] To evaluate the Piezopen for DNA delivery, live human skin samples were used ex vivo. Intradermal (ID) injections were administered on Day 0, using either distilled water alone (Naive; UltraPure™ DNase / RNase-Free Distilled Water from ThermoFisher Scientific, Catalog #10977015) or a solution containing 100 pg of gWiz-Euciferase DNA (DNA-Euc; Aldevron, Catalog #5000) in 50 pF of distilled water. These injections were performed with and without electroporation (EP), using either the Piezopen or the BTX device. After 24 hours, luciferin (Revvity, Catalog #122799) was injected at the corresponding sites to measure DNA-Euc expression, with bioluminescence signals tracked using an in vivo imaging system (IVIS) for 24 hours. The BTX EP protocol included the following sequence: one pulse at 450 V for 0.05 ms with a 0.2 ms interval, one pulse at 450 V for 0.05 ms with a 50 ms interval, and eight pulses at 110 V for 10 ms at a 20 ms interval.

[0207] Microneedle Injections

[0208] On Day 0, DNA-Luc was administered to ex vivo human skin samples via vertical intradermal (VID) injections. Electroporation (EP) was then applied using both the Piezopen and BTX devices. Each sample received 100 pg of DNA-Luc prepared in 50 pL of water. DNA was prepared and stored in a -20°C cryo-box until use. After DNA delivery, samples were incubated for 24 hours prior to imaging. For in vivo imaging system (IVIS) analysis, luciferin was injected intradermally at each injection site at a dose of 150 mg / kg, followed by a 15-minute wait to ensure adequate distribution before imaging.

[0209] Preparation of Luciferin for IVIS Imaging

[0210] A stock solution of luciferin was prepared at a concentration of 30 mg / mL in water, aliquoted, and stored at -20°C. Before imaging, the required volume of luciferin was thawed, and a fresh working solution was diluted to 15 mg / mL in water. Each injection site received a 50 pL intradermal (ID) injection of luciferin 15 minutes prior to imaging to ensure proper distribution and optimal signal capture during IVIS imaging.

[0211] Use of Living Image Software on IVIS to Capture and Analyze Luciferase Signals Luciferase imaging with the IVIS system was performed and radiant efficiency was calculated using Living Image software, with careful attention given to maintaining a consistent region of interest (ROI) area across all groups for precise comparison. The ROI was adjusted to fully encompass the fluorescence expression for each group, starting with the experimental group and then applied to the control group. Images were saved with both raw fluorescence intensity and calculated radiant efficiency for future analysis and reference.

[0212] Human Skin Model

[0213] The human skin samples used were NativeSkin® models obtained from Genoskin, which include donated human skin biopsies preserved in a nourishing matrix with a culture medium to sustain tissue viability for up to seven days post-surgery. For this study, NativeSkin® models of 20 mm in diameter were used. Each sample was placed in a well of a 6-well plate, and the media was changed daily to maintain tissue health throughout the experiment. These models were used to evaluate intradermal (ID) and intracellular delivery methods under standardized conditions.

[0214] Experimental Groups

[0215] This study included the following experimental groups: (1) Naive (water), (2) ID injection of DNA-Luc, (3) VID injection of DNA-Luc with varying needle lengths (1.0 mm, 1.1 mm, 1.25 mm, and 1.3 mm), (4) VID injection of DNA-Luc followed by Piezopen electroporation (VIDEP Separate; VIDEP S), and (5) ID injection of DNA-Luc followed by BTX electroporation. The numbers specified in figure captions represented technical replicates, indicating the number of regions of interest (ROI) analyzed for each group.

[0216] Results

[0217] It was observed that VID injections produced comparable expression levels to intradermal (ID) injection using the Mantoux method one day after administration (Figures 6A and 6B). Furthermore, VID injection of DNA-Luc followed by application of the Piezopen device led to a significant increase in gene expression — approximately 1000-fold higher — compared to VID injection alone (Figures 6C and 6D). This increase in gene expression demonstrates successful intracellular DNA delivery in an ex vivo human model, supporting the potential of VID injection with Piezopen to be safely and effectively translated into clinical applications.

[0218] A range of commercially available needles and needle lengths was screened to determine the most effective setup for vertical intradermal (VID) injections. It was observed that needle lengths between 1.0 mm and 1.4 mm were effective for VID injections, as evidenced by the back pressure encountered during injection and the characteristic bleb formation (Figure 7A). These needle lengths were validated in human skin models, showing similar levels of expression 24 hours post-delivery when compared to the standard Mantoux method without electroporation (EP) (Figures 7B and 7C). For subsequent experiments, a 1.0 mm needle was selected.

[0219] Further, the efficacy of VID injection followed by electroporation (EP) using the Piezopen device was assessed in human skin samples. Initial results indicated that the Piezopen device achieved approximately 1.5 orders of magnitude higher gene expression compared to injection alone (Figures 7B and 7C), confirming successful intracellular DNA delivery ex vivo and supporting its potential for clinical application. In comparative analyses, Piezopen EP produced approximately 1 order of magnitude higher expression than BTX EP (Figures 7D, 8A and 8B). Overall, Piezopen EP significantly increased gene expression compared to the commercially available BTX electroporator and control conditions (Naive and VID injection alone). Total flux observed on Day 1 was as follows: Naive: 1.5xl04, ID: 5.9xl05, VID: 3.9xl05, ID BTX: 3.2xl06, and VIDEP Separate: 1.4xl07(Figures 8A and 8B).

[0220] Conclusions

[0221] The findings demonstrate that drug delivery needle length is important for successful intradermal (ID) injection ex vivo. Both vertical intradermal (VID) and ID injections produced comparable gene expression levels, validating the VID injection approach. Additionally, VID injection followed by electroporation (EP) using the Piezopen device demonstrated superior efficacy compared to ID injection alone, followed by the commercially available BTX EP, highlighting the advanced efficacy of Piezopen over the currently existing models. This study confirmed that VID injection of DNA is feasible and can be effectively integrated with EP. The Piezopen device’s EP performance was shown to surpass that of the BTX electroporator ex vivo, supporting its potential to replace existing EP systems.

[0222] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0223] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific instances of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A microelectrode array comprising: an array of biocompatible and conductive microelectrodes comprising an opening for at least one needle therein; wherein a syringe comprising a container for holding at least one agent can be coupled to the at least one needle for delivery of the at least one agent.

2. The microelectrode array of claim 1, further comprising a base from which the array of biocompatible and conductive microelectrodes extends.

3. The microelectrode array of any one of claims 1-2, wherein the biocompatible and conductive electrodes are formed of a metal; or wherein the biocompatible and conductive electrodes are formed of a mixture of epoxy and graphite, a mixture of glass and platinum, or a conductive ceramic.

4. The microelectrode array of claim 3, wherein the metal is stainless steel.

5. The microelectrode array of claim 1, wherein the biocompatible and conductive microelectrodes of the array extend from one or more metal plates.

6. The microelectrode array of claim 5, wherein a linear array of the biocompatible and conductive microelectrodes extends from one edge of each of the one or more metal plates.

7. The microelectrode array of claim 5 or 6, wherein the one or more plates are parallel to each other and spaced apart from one another.

8. The microelectrode array of any one of claims 1-2, wherein the biocompatible and conductive microelectrodes of the array extend from a single metal plate, and the array is a two- dimensional array.

9. The microelectrode array of any one of claims 1-2, wherein the biocompatible and conductive microelectrodes of the array extend from one or more metal plates and the array is a two-dimensional array.

10. The microelectrode array of any one of claims 1-2, wherein the biocompatible and conductive microelectrodes of the array extend from at least one non-electrically conductive plate and wherein electrical connections are provided between the biocompatible and conductive microelectrodes.

11. The microelectrode array of claim 10, wherein the electrical connections are located on a surface of the at least one non-electrically conductive plate.

12. The microelectrode array of claim 10, wherein the electrical connections cross from a first side of the at least one non-electrically conductive plate to an opposed second side of the plate through holes in the at least one non-electrically conductive plate.

13. The microelectrode array of claim 1, wherein the biocompatible and conductive microelectrodes of the array have an area between about 1 mm2and 50 cm2or about 1 mm2and 25 cm2.

14. The microelectrode array of claim 1, wherein the array of biocompatible and conductive microelectrodes comprises from: (1) about 2 to 3000 microelectrodes, about 2 to 2000 microelectrodes, or about 2 to 1000 microelectrodes; or (2) at least: about 25 microelectrodes, about 50 microelectrodes, about 100 microelectrodes, about 150 microelectrodes, about 200 microelectrodes, about 250 microelectrodes, about 300 microelectrodes, about 350 microelectrodes, about 400 microelectrodes, about 450 microelectrodes, about 500 microelectrodes, about 550 microelectrodes, about 600 microelectrodes, about 650 microelectrodes, about 700 microelectrodes, about 750 microelectrodes, about 800 microelectrodes, about 850 microelectrodes, about 900 microelectrodes, about 950 microelectrodes, about 1000 microelectrodes, about 1500 microelectrodes, about 2000 microelectrodes, about 2500 microelectrodes, or about 3000 microelectrodes.

15. The microelectrode array of claim 1, wherein the biocompatible and conductive microelectrodes of the array each independently have a length from 10 pm to 8 mm; or wherein the biocompatible and conductive microelectrodes of the array each independently have a length of at least about 500 pm, 600 pm, 650 pm, 700 pm, 750 pm, 800 pm, 900 pm, 950 pm, 1000 pm, 1050 pm, 1100 pm, 1150 pm, 1200 pm, 1250 pm, 1300 pm, 1350 pm, 1400 pm, 1450 pm, or 1500 pm.

16. The microelectrode array of claim 1, wherein the biocompatible and conductive microelectrodes of the array have: a spacing in the array between about 0.1 mm and 3 mm, between about 0.2 mm and 2.5 mm, between about 0.3 mm and 2 mm, between about 0.5 mm and 1.5 mm, between about 0.5 mm and 1.0 mm, between about 0.5 mm and 0.9 mm, between about 0.5 mm and 0.8 mm, between about 0.5 mm and 0.7 mm, or between about 0.5 mm and 0.6 mm; and / or the microelectrodes of the array are arranged in a rectangular, square, triangular, or circular orientation.

17. The microelectrode array of claim 1, wherein the at least one needle is a hypodermic needle, a hollow microneedle, an insulin syringe needle, or a pen needle.

18. The microelectrode array of claim 1, wherein the microelectrode array further comprises a patch of microneedles which are optionally coated and / or dissolvable.

19. A device comprising: a piezoelectric pulse generator; an array of biocompatible and conductive microelectrodes electrically coupled to the piezoelectric pulse generator; and a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses; wherein the array of biocompatible and conductive microelectrodes comprises an opening for insertion of at least one needle therein for delivery of at least one agent; wherein a syringe comprising a container for holding the at least one agent can be coupled to the at least one needle and the at least one agent can be administered into a biological tissue by actuation of the syringe; wherein insertion of the array of biocompatible and conductive microelectrodes into the biological tissue and activation of the piezoelectric pulse generator generates the one or more electrical pulses through the array of biocompatible and conductive microelectrodes to electroporate cells in the biological tissue to permit administration of the at least one agent into the electroporated cells.

20. The device of claim 19, the device further comprising at least one needle.

21. The device of claim 20, wherein the at least one needle is a hypodermic needle, a hollow microneedle, an insulin syringe needle, or a pen needle.

22. The device of any one of claims 19-21, the device further comprising a syringe.

23. The device of any one of claims 19-21, further comprising (i) a base from which the array of biocompatible and conductive microelectrodes extends, and (ii) a housing which contains the piezoelectric pulse generator and the switch.

24. The device of any one of claims 19-21, further comprising (i) a base from which the array of biocompatible and conductive microelectrodes extends, and (ii) a housing which contains the base, the piezoelectric pulse generator, and the switch.

25. The device of any one of claims 19-21, the device further comprising the at least one agent.

26. The device of claim 25, wherein the at least one agent is selected from the group consisting of a nucleic acid (such as DNA and / or RNA, nucleic acid encoding a vaccine, nucleic acid encoding a therapeutic protein, nucleic acid encoding a hormone, or a nucleic acid encoding a monoclonal antibody), a DNA-plasmid, a minicircle DNA plasmid, an mRNA, a selfamplifying RNA, a circular RNA, a transfer RNA, a chemotherapeutic agent, a biologic, a prophylactic, a DNA-launched self- amplifying RNA, a DNA-launched virus, a linear DNA, and combinations thereof.

27. The device of claim 24, wherein the at least one agent is a therapeutic.

28. The device of claim 27, wherein the vaccine is an RNA therapeutic or a DNA therapeutic.

29. The device of any one of claims 19-21, wherein the biocompatible and conductive microelectrodes are formed of a metal; or wherein the biocompatible and conductive electrodes are formed of a mixture of epoxy and graphite, a mixture of glass and platinum, or a conductive ceramic.

30. The device of claim 29, wherein the metal is stainless steel.

31. The device of any one of claims 19-21, wherein the biocompatible and conductive microelectrodes of the array extend from one or more metal plates which are configured to conduct the one or more electrical pulses from the piezoelectric pulse generator to the microelectrodes of the array.

32. The device of claim 31 , wherein a linear array of the biocompatible and conductive microelectrodes extends from one edge of each of the one or more metal plates.

33. The device of claim 31 , wherein the one or more plates are parallel to each other and spaced apart from one another.

34. The device of any one of claims 19-21, wherein the biocompatible and conductive microelectrodes of the array extend from a single metal plate, and the array is a two-dimensional array.

35. The device of any one of claims 19-21, wherein the biocompatible and conductive microelectrodes of the array extend from one or more metal plates and the array is a two- dimensional array.

36. The device of any one of claims 19-21, wherein the biocompatible and conductive microelectrodes of the array extend from at least one non-electrically conductive plate and wherein electrical connections are provided between the biocompatible and conductive microelectrodes and configured to conduct the one or more electrical pulses from the piezoelectric pulse generator to the microelectrodes of the array.

37. The device of claim 36, wherein the electrical connections are located on a surface of the at least one non-electrically conductive plate.

38. The device of claim 36, wherein the electrical connections cross from a first side of the at least one non-electrically conductive plate to an opposed second side of the plate through holes in the at least one non-electrically conductive plate.

39. The device of any one of claims 19-21, wherein the piezoelectric pulse generator comprises a piezoelectric crystal selected from the group consisting of lead zirconate titanate (PZT), silicon nitride, barium titanate, quartz, zinc oxide, sodium tungstate, sodium potassiumtartrate, tourmaline, lithium niobate, gallium arsenide, aluminum nitride, and combinations thereof.

40. The device of any one of claims 19-21, wherein the switch triggers a spring -latch hammer mechanism configured to strike a surface of the piezoelectric crystal of the piezoelectric pulse generator.

41. The device of claim 40, further comprising a pin disposed between the spring-latch hammer mechanism and the piezoelectric crystal.

42. The device of any one of claims 19-21, wherein the switch is a toggle switch with a latch configured to release a hammer, configured to strike a surface of the piezoelectric crystal of the piezoelectric pulse generator effective.

43. The device of claim 42, further comprising a pin disposed between the latch and the piezoelectric crystal.

44. The device of any one of claims 19-43, further comprising a casing for the piezoelectric crystal; and wherein electrical connections comprised of a first electrode and a second electrode extend from the casing.

45. The device of claim 44, further comprising a cartridge that contains the array of biocompatible and conductive microelectrodes and comprises a first receptacle for mating engagement with the first electrode and a second receptacle for mating engagement with the second electrode, the first and second receptacles are in electrical communication with the microelectrodes of the array.

46. The device of any one of claims 19-21, wherein the microelectrodes of the array are arranged to be inserted into the biological tissue having an area between about 1 mm2and 50 cm2or about 1 mm2and 25 cm2.

47. The device of any one of claims 19-21, wherein the array of biocompatible and conductive microelectrodes comprises from: (1) about 2 to 3000 microelectrodes, about 2 to 2000 microelectrodes, or about 2 to 1000 microelectrodes; or (2) at least: about 25 microelectrodes, about 50 microelectrodes, about 100 microelectrodes, about 150 microelectrodes, about 200 microelectrodes, about 250 microelectrodes, about 300 microelectrodes, about 350 microelectrodes, about 400 microelectrodes, about 450 microelectrodes, about 500 microelectrodes, about 550 microelectrodes, about 600 microelectrodes, about 650 microelectrodes, about 700 microelectrodes, about 750 microelectrodes, about 800 microelectrodes, about 850 microelectrodes, about 900 microelectrodes, about 950 microelectrodes, about 1000 microelectrodes, about 1500 microelectrodes, about 2000 microelectrodes, about 2500 microelectrodes, or about 3000 microelectrodes.

48. The device of any one of claims 19-21, wherein the microelectrodes of the array each independently have a length from 10 pm to 8 mm; or wherein the biocompatible and conductive microelectrodes of the array each independently have a length of at least about 500 pm, 600 pm, 650 pm, 700 pm, 750 pm, 800 pm, 900 pm, 950 pm, 1000 pm, 1050 pm, 1100 pm, 1150 pm, 1200 pm, 1250 pm, 1300 pm, 1350 pm, 1400 pm, 1450 pm, or 1500 pm.

49. The device of any one of claims 19-21, wherein the array of biocompatible and conductive microelectrodes is replaceable; and / or wherein the piezoelectric pulse generator and the switch are each independently single-use or are reusable.

50. The device of any one of claims 19-21, wherein the one or more electrical pulses have a peak voltage absolute value of between about 10 V and 35,000 V, 10 V and 35,000 V, 10 V and 30,000 V, 10 V and 20,000 V, 10 V and 10,000 V, 20,000 V and 30,000 V, between about 50 V and 5,000 V, between about 100 V and 1,000 V, or between about 200 V and 500 V when measured in air; or wherein the one or more electrical pulses have a peak voltage absolute value of between about 100 V and 1000 V, about 100 V and 900 V, about 100 V and 800 V, about 100 V and 700 V, about 100 V and 600 V, about 100 V and 500 V, about 100 V and 400 V, about 100 V and 300 V, about 100 V and 200 V, about 200 V and 400 V, between about 200 V and 350 V, between about 200 V and 300 V, or between about 200 V and 250 V when measured in a tissue.

51. The device of any one of claims 19-21, wherein the one or more electrical pulses have a ratio of absolute value of peak voltage to absolute value of peak-to-peak voltage between about 0.1 and 10, between about 0.3 and 5, or between about 0.5 and 2.

52. The device of any one of claims 19-21, wherein the one or more electrical pulses have a peak current absolute value between about 0.001 A and 1,000 A, between about 0.01 A and 500 A, between about 0.1 A and 100 A, or between about 1 A and 50 A.

53. The device of any one of claims 19-21, wherein the one or more electrical pulses have a peak static voltage absolute value of between about 100 V and 35,000 V, between about 1,000 V and 30,000 V, or between about 15,000 V and 35,000 V when measured in air.

54. The device of any one of claims 19-21, wherein the one or more electrical pulses produce an electric field strength of between about 100 V / cm and 30,000 V / cm, between about 200 V / cm and 10,000 V / cm, between about 300 V / cm and 5,000 V / cm, or between about 500 V / cm and 3,500 V / cm.

55. The device of any one of claims 19-21, wherein the one or more electrical pulses generated have an initial pulse length of between about 1 ps and 10,000 ps, between about 1 ps and 1,000 ps, between about 1 ps and 100 ps, between about 3 ps and 100 ps, between about 5 ps and 50 ps , or between about 10 ps and 30 ps.

56. The device of any one of claims 19-21, wherein the one or more electrical pulses generated have a ratio of initial pulse length to total pulse length between about 1.5 and 100, between about 2 and 50, or between about 3 and 20.

57. The device of any one of claims 19-21, wherein the microelectrodes of the array have: a spacing in the array between about 0.1 mm and 3 mm, between about 0.2 mm and 2.5 mm, between about 0.3 mm and 2 mm, between about 0.5 mm and 1.5 mm, between about 0.5 mm and 1.0 mm, between about 0.5 mm and 0.9 mm, between about 0.5 mm and 0.8 mm, between about 0.5 mm and 0.7 mm, or between about 0.5 mm and 0.6 mm; and / or the microelectrodes of the array are arranged in a rectangular, square, triangular, or circular orientation.

58. The device of any one of claims 19-21, wherein the device further comprises a patch or a coating, which are optionally dissolvable59. A method of delivering an agent into or across a biological tissue, the method comprising the steps of:(a) positioning a device adjacent to a biological tissue site, the device comprising: a piezoelectric pulse generator; an array of biocompatible and conductive microelectrodes electrically coupled to the piezoelectric pulse generator; a switch coupled to the piezoelectric pulse generator for activating the piezoelectric pulse generator to generate one or more electrical pulses; a syringe comprising a container holding at least one agent; wherein the array of biocompatible and conductive microelectrodes comprises at least one needle for delivery of the at least one agent embedded therein; wherein the syringe is coupled to the at least one needle for administering the at least one agent into a biological tissue by actuation of the syringe;(b) contacting the array of biocompatible and conductive microelectrodes comprising the at least one needle embedded therein into the target biological tissue site;(c) activating the switch to generate and deliver one or more electrical pulses through the array of biocompatible and conductive microelectrodes into the target biological tissue site to electroporate cells at the target biological tissue site; and(d) administering the at least one agent into the target biological tissue site through the at least one needle by actuation of the syringe.

60. The method of claim 59, wherein step (c) and step (d) are performed concurrently.

61. The method of claim 59, wherein step (d) is performed following step (c).

62. The method of claim 59, wherein step (c) is performed following step (d).

63. The method of any one of claims 59-62, wherein step (c) is repeated at least once.

64. The method of any one of claims 59-62, wherein the target biological tissue site comprises mammalian skin.

65. The method of any one of claims 59-62, wherein the target biological tissue site is mammalian skin dermis.

66. The method of any one of claims 59-62, wherein the target biological tissue site is mammalian skin epidermis.

67. The method of any one of claims 59-62, wherein the target biological tissue site is or comprises a mucosal membrane.

68. The method of any one of claims 59-62, wherein the one or more electrical pulses have a peak voltage absolute value between about 100 V and 1000 V, a peak current absolute value between about 0.001 A and 50 A, a peak static voltage absolute value between about 15,000 V and 35,000 V, an initial pulse length of between about 1 ps and 100 ps, or a combination thereof.

69. The method of any one of claims 59-62, wherein the at least one agent is selected from the group consisting of a nucleic acid (such as DNA and / or RNA, nucleic acid encoding a vaccine, nucleic acid encoding a therapeutic protein, nucleic acid encoding a hormone, or a nucleic acid encoding a monoclonal antibody), a DNA-plasmid, a minicircle DNA plasmid, an mRNA, a self- amplifying RNA, a circular RNA, a transfer RNA, a chemotherapeutic agent, a biologic, a prophylactic, a DNA-launched self-amplifying RNA, a DNA-launched virus, a linear DNA, and combinations thereof.

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